Process for producing material for smoking

ABSTRACT

A process is disclosed for producing sheet-like material for smoking which includes the steps of: culturing a fragment of a plant body of the genus Nicotiana in a liquid medium under aerobic conditions to derive tobacco cells suspended in the liquid, filtering the cultured broth containing the tobacco cells or the same mixed with fibrous materials and/or inorganic materials through a gauze of 50-120 mesh or filter cloth, so as to form a sheet-like cake having a thickness corresponding to dried weight above 50 g./m2 and drying said sheet-like cake. Additionally, a process is disclosed for producing rod-like material for smoking which includes the steps of: culturing a fragment of a plant body of the genus Nicotiana in a liquid medium under aerobic conditions, to derive tobacco cells suspended in the liquid, separating the tobacco cells from the cultured broth, mixing the tobacco cells with tobacco leaf substances and/or fibrous materials and/or inorganic materials to form a paste with a moisture content of 80-93 percent, casting the paste into a rod-shape and freeze-drying said rod-shaped paste. The produce has excellent physical properties and the cigarettes made thereof have light and good organoleptic properties.

United States Patent [1 1 Tamaki et al.

[ 51 Jan. 16, 1973 l 54] PROCESS FOR PRODUCING MATERIAL FOR SMOKING [75]Inventors: Einosuke Tamaki; Masao Kobari, both of Tokyo; FumihiroMiyanowaki; Kunio Kato, both of Yokohama; K0 Nishida; Yukio Shimizu,both of Tokyo, all of Japan [73] Assignee: The Japan MonopolyCorporation,

Tokyo, Japan 22 Filed: Sept. 14,1971

211 Appl. No.: 180,353

[30] Foreign Application Priority Data 3,628,287 12/1971 Stabaetal..47/58 Primary ExaminerRobert E. Bagwill Attorney-Toren and McGeady{57] ABSTRACT A process is disclosed for producing sheet-like materialfor smoking which includes the steps of: culturing a fragment of a plantbody of the genus Nicotiana in a liquid medium under aerobic conditionsto derive tobacco cells suspended in the liquid, filtering the culturedbroth containing the tobacco cells or.the same mixed with fibrousmaterials and/or inorganic materials through a gauze of 50-120 mesh orfilter cloth, so as to form a sheet-like cake having a thicknesscorresponding to dried weight above 50 g./m and drying said sheet-likecake. Additionally, a process is disclosed for producing rod-likematerial for smoking which includes the steps of: culturing a fragmentof a plant body of the genus Nicotiana in a liquid medium under aerobicconditions, to derive tobacco cells suspended in the liquid, separatingthe tobacco cells from the cultured broth, mixing the tobacco cells withtobacco leaf substances and/or fibrous materials and/or inorganicmaterials to form a paste with a moisture content of 80-93 percent,casting the paste into a rod-shape and freeze-drying said rod-shapedpaste. The produce has excellent physical properties and the cigarettesmade thereof have light and good organoleptic properties.

20 Claims, 2 Drawing Figures PATENTEDJM 1 I97 710.805

SHEET 1 [1F 2 C15 do ,3 g i 9 S (I) m g 10 8 F 3 O l I l l I I I z. 5 s7 a 91011 Time (days) PROCESS FOR PRODUCING MATERIAL FOR SMOKING Thepresent invention relates to a process for producing material forsmoking using the tissue-culture of plants of the genus Nicotiana as rawmaterials.

The term callus in the present specification and claims refers to anamorphous lump of cells which have lost organ-forming capacity, which isformed when a fragment of the plant body is tissue-cultured on a solidmedium, and which shows an external form resembling the agglutinationtissue of the plant body. The term tobacco cells refers to a fine flockydispersion of the cells formed when pieces of the said callus arefurther inoculated and tissue-cultured in a liquid medium under aerobicconditions. The liquid containing the tobacco cells is represented bythe term cell suspension or cultured broth."

Smoking tobacco has hitherto been produced by a process consisting of along term cultivation step of a plant of the genus Nicotiana in thefield and a complex operation step for cigarette-making using theharvested leaf of said plant. However, said cultivation step of theplant is completely at the mercy of landand climaticconditions, andtherefore the kind, quantity, quality, and the like, of the tobacco leafwhich is the main raw material of cigarettes have not necessarily beenfree from the influence of the above-mentioned natural conditions.

The plant body is generally composed of innumerable cells, which formthe tissues and organs of the plant and perform living phenomenathereby. It was found lately that a complete plant body can be growndirectly from any voluntarily taken-up cells of the plant body byso-called tissue-culturing of said cells, and such tissueculture wasapplied for studies in the improvement of plant breeding by selectingcells having excellent plant genetic characteristics. Further it isknown that socalled callus can be formed on a solid medium by means ofsuch tissue-culture, while a suspension of fine cells of the plant canbe obtained by tissue-culturing said callus in liquid medium underaerobic conditions.

We have investigated the above-mentioned tissueculture of plants ofgenus Nicotiana, and found that the tobacco cells derived from thecallus and propagated in cultured broth can be made readily intosheet-like or rod-like materials for smoking.

An object of the invention is to provide a simple and facile process forproducing material for smoking.

Another object of the invention is to provide an industrial process forproducing material for smoking, which is not influenced by landandsoil-conditions or climatic conditions.

Still another object of the invention is to provide a process forproducing material for smoking containing little or no nicotine andhaving excellent physical and organoleptic properties.

Other objects of the invention will readily be understood from thefollowing explanation.

Referring now to the drawings,

P16. 1 is a graph showing the increase of formed tobacco cells and thedecrease of remaining sugar in the cultured broth, with the lapse oftime, when the cells of Nicotiana tabacum var. bright yellow aretissuecultured under aerobic condition, using liquid media in which thecontent of KH PO is varied FIG. 2 is a schematic flowsheet illustratingthe process for producing sheet-like material for smoking from tobaccocells propagated in the cultured broth.

According to the present invention, the following plants of the variousspecies belonging, to genus Nicotiana may be used for tissue-culturecultural varieties such as N. tabacum var. bright yellow, N. 1. var.xanthi ova, N. t. var. burley 2l, N. 1. var. enshu, N. 1. var.matsukawa, N. t. var. nanbu, N. 1. var. shirodaruma, N. 1. var. suifu,N. t. xanthi yaka, N. velutina, N. rustica, and the like, and wildspecies such as N. glutinosa, N. acuminata, N. afflm's, N. amplexcaulis,N. arentsii, N. bigelavii, N. clevelandii, N. codifolia, N. debneyi, N.exclesior, N. fragrans, N. glauca, N. gossei, N. ingulba, N. knightianaN. langsdorfii, N. miersii, N. repqnda, N. multivalvis, N. occidentalisvar. obliqua, N. rosulata, N. occidentalis var. 0001', N. pauciflora, N.quadrivalvis, N. raimondii, N. simlans, N. solanifolia, N. stenocarpa,N. sylvestris, and the like.

These plants of genus Nicotiana have physiological properties moresuitable for tissueculture in comparison with other kinds of plantbodies, and many studies on tissue-culture using the plants of genusNicotiana have hitherto been reported. Further, several suitablemedium-compositions for tissue-culture have been mentioned in theliterature, for example, so-called Whites medium (1943), Hellers medium(1953), Murashige and Skoogs medium (1962) and Linsmaier and Skoogsmedium (1965).

The said known media consist of inorganic substances and other minuteelements which have hitherto been used in the medium for thewater-culture method for plants, such as, saccharides, auxins(growthpromoting substance), cytokinin s, vitamins and amino acids. Inparticular the following substances are used in these media: inorganicsalts such as potassium chloride, calcium chloride, potassium nitrate,calcium nitrate, sodium nitrate, ammonium nitrate, sodium sulfate,magnesium sulfate, potassium phosphate, sodium phosphate, ferricchloride, ferric sulfate, Na -EDTA (Na Ethylenediamine tetra-aceticacid), manganese sulfate, zinc sulfate, boric acid, potassium iodide,copper sulfate, sodium molybdate, aluminum chloride and cobalt chloride,saccharides such as sucrose, glucose, fructose and mannose, auxins suchas 2,4- dichlorophenoxy-acetic acid, a-naphthaleneacetic acid andindol3-acetic acid, cytokinins such as kinetin, vitamins such as thiaminhydrochloride, pyridoxin hydrochloride, nicotinic acid, myo-inositol andbiotin, and amino acids such as glycine.

We have examined the compositions of said conventional media fortissue-culture and disclosed the following subjects:

l. The yield of tobacco cells in the tissue-cultures using the knownmedia was highest when the Linsmaier and Skoogs medium was adopted.

2. It has a favorable effect on the yield of tobacco cells to usequantities of phosphate from twice to thrice as much as that of theLinsmaier and Skoogs medium which contains the largest quantity ofphosphate among the said known media, i.e., 340-510 mg./liter, as shownin FIG. 1, where the solid-line designated with PiX3 is the curveindicating the dried weight of tobacco cells formed when a quantity ofphosphate thrice as much as that of the medium was used, and thesolid-line designated with PiXl is the similar curve when a quantity ofphosphate equal to that of the medium was used, while the dotted-lineswith the same designations are the curves of remaining sugar in thecultured broth corresponding to said solid-lines, respectively.

3. In the Linsmaier and Skoogs medium, myo-inositol can be replaced withthiamin hydrochloride.

From the above, an example of the suitable medium for tissue-cultureused in the process of the invention is shown below:

TABLE 1 NH,NO, 1,650 mgJliter KNO; 1,900 CaClflH O 440 MgSO,-7H.,O 370KH PO 340 Na -EDTA 37.3 FcSO,-7H.,O 27.8 H BO 6.2 MnSO,'4H,O 22.3ZnSO,-4H O 8.6 Kl 0.83 Na MoO,'2H,O 0.25 CuSO,-5H O 0.025 CoCl -6H O0.025 Sucrose 40,000 2,4-dichlorophenoxy-acetic acid 0.2 Thiaminhydrochloride l.0 Kinetin 0.2 pH (after sterilization) 5.0-6.2

As regards the use of the tissue-culture from the plant body of thegenus Nicotiana in the present invention, for example, fragments of theleaf, stern, root, flower, seed or other organs or tissues of the plantare washed, surface-sterilized, placed on a sterile agar medium fortissue-culture which has a composition as described in Table l and iscontained in an Erlenmeyers flask plugged with cotton wool, and areincubated at 25-35 C. Said fragments of organs or tissues swell andcallus is derived in 4-5 weeks.

Such callus can be gradually made purer by means of repeating the abovesolid medium-culturing, that is, by inoculating fresh solid medium inturn with small pieces of callus which have been cutoff from callusformed in the previous solid medium-culturing.

The callus thus reformed and refined on the solid medium is theninoculated into a liquid medium having the same composition as that ofthe solid medium but without agar, and cultured on a shaker at atemperature of 25-3 5 C for 2-3 weeks. The inoculum is about 3 g. ofcallus by fresh weight to 100 ml. of liquid medium, and the calluspropagates in the culture liquid as a flocky suspension, that is, astobacco cells." These tobacco cells become finer and are more rapidlyformed when the above shake-culture in liquid medium is repeated,inoculating fresh liquid medium in turn with a portion of the previouslycultured broth containing tobacco cells.

The cell suspension obtained in the shake-culture is, after beingscaled-up by turns as described above, inoculated into a liquid mediumof the same composition set in a fermenter made of stainless steel, andcultured with aeration while being agitated gently. The inoculum is onetenth of the quantity of the whole medium, and intensive agitation isunfavorable as the membranes of tobacco cells maybe broken. Thequantities of air required to aerate are about 0.5-2.0 liters/liter ofmedium/minute and the culturing period is within a week.

Tobacco cells, thus cultured and propagated in a large quantity by theaeration culture with stirring, can be separated from the cultured brothby filtration or centrifugation, without the cell membranes beingbroken. The separated tobacco cell collection contains 90-95 percent(W.B.) of moisture, and the yield of 0 dried weight of these tobaccocells is 50-65 percent of the sugar consumed in the cultured broth andamounts to 15-20 g. per liter of the medium (in condition of usingbatchwise cultivation). The single cell constituting these tobacco cellsis an ellipsoid having a longer diameter of about 50-200 p. and ashorter diameter of about 30-50 ;1..

ln connection with the above explanation, it is observed that it takesat least 4-5 weeks of culture on agar medium, 2-3 weeks ofliquidshake-culture and about a week of aeration-liquid culture with stirring,totaling to a long term of 7-9 weeks, to obtain tobacco cells asmaterials for smoking, and moreover such a total culture term isremarkably prolonged when both the solid mediumand liquidmedium-culturings are repeated as described above. However, 1 once acultured broth where tobacco cells have been propagated in homogeneoussuspension is obtained in the aerationculture with stirring, additionalcultured broth can be obtained within a short period by adopting asemi-continuous process, where a portion of the cultured broth is takenout to leave the other portion of said broth in the fermenter and freshsterile medium is supplemented to the remaining broth so as to carry onthe culturing of the tobacco cells again. For example, when about a halfof the volume of the cultured broth is taken out from the fermenter andaeration-culture is carried on after supplying the fermenter with freshmedium, the subsequent logarithmic growth-phase of the tobacco cells inthe culturing will be approximately completed within a day or two, andconsequently about a half of the approximately finished cultured broth(containing tobacco cells of 13-15 g. by dried weight per liter) can beobtained everyday or every other day by employing the semi-continuousprocess. Furthermore, the cultured broth taken out from the fermenter asdescribed above contains a relatively large quantity of remaining sugar,and consequently it is advantageous to return the filtrate of thetaken-out cultured broth to the initial fermenter, after supplementingwith fresh medium-constituents and sterilizing the same, so as tore-utilize the filtrate.

According to the present invention, firstly, the tobacco cells obtainedby tissue-culture of the plants of genus Nicotiana are made into asheet-like material for smoking. There have hitherto been severalmethods for producing the sheet-like materials for smoking, for example,extrusion, slurry, paper-making and microflaking. However, each of theseconventional processes are composed of several complex steps.

Now we have discovered that sheet-like material for smoking havingexcellent physical and organoleptic properties can be obtained withoutany complex operation, by using tobacco cells as the major rawmaterials. That is, fresh tobacco cells having a specific type of ofviscidity and consequently sheet-like material for smoking can beobtained by filtering the cultured broth containing such tobacco cellsor the same mixed with the fibrous materials and/or inorganic substancesto form a cake (filtration residue) and drying this cake as it is. Theabove process is carried out without using any of the binders andwater-proofing agents which have been generally indispensable in theconventional processes for producing the sheet-like material.

According to the present invention, a cultured broth containing thetobacco cells, or the same mixed with the fibrous materials and/orinorganic materials which do not damage the smoking characteristics ofthe final products therefrom, are filtered through a gauze of 50-120mesh or filter cloth, so as to form a filtration cake with a thicknesscorresponding to a dried weight above 50 g./m If the dried weight/m ofthe cake is below 50 g., the thickness of the cake will not be uniformwhich causes inconveniences in the subsequent treatments. It isdesirable that the moisture content of the cake be reduced to below 95percent (W.B.), by using a sucker-type filter. Such filtration by meansof suction can be carried out within an extremely short time, and therate of recovery of tobacco cells from the cultured broth is about 95-98percent.

Suitable as the fibrous material to be mixed with the cultured broth inthe present invention, are vegetable fiber such as wood pulp, hemp,bagasse, beet, and tobacco vein, and inorganic fiber such as glassfibers, and carbon fiber. Silica, alumina, asbestos, kieselguhr, talc,calcium carbonate and zeolite maybe used as the inorganic materials.These fibrous materials and inorganic materials are effective not onlyas a filter medium in the above filtration but also to improve thephysical properties, especially the strength and filling value, of thesheet-like material made therefrom. The amount of the fibrous andinorganic materials to be mixed with the cultured broth are below 30percent based on the dried weight of the tobacco cells containedtherein.

ln the present invention, tobacco powder, tobacco dust, vein (these willbe represented by the term tobacco leaf substances hereafter in thespecification and claims), and/or the supplementary agents such asaromatics, burning agents, sweetenings, and hydroscopic agents, may bemade to adhere to the above filtration cake by spreading the samethereon and pressing the cake gently between rolls, in the same way asin the conventional process for producing sheet-like materials forsmoking. However, alternatively the aromatics, sweetenings andhydroscopic agents among said supplementary agents may be laterincorporated into finished (dried) sheet-like material in the invention. As the aromatics described above, essential oils such ascascarilla oil, bergamot oil, iris oil, extracts such as benzointincture, tobacco extract, fruit extract, and perfumeries such ascoumarin, vanillin, and menthol, may be used. Active carbon, sodiumfumarate, potassium fumarate, and the like, maybe used as burningagents, while sucrose, glycerol, propyleneglycol, sorbitol, and thelike, maybe used as sweetenings or hydroscopic agents. The quantities ofthe tobacco leaf substances to be made to adhere to the cake are belowthe value of the dried weight of the tobacco cells contained in thecake. The quantities of the above supplementary agents to be added arerather small, and therefore addition of these agents have littleinfluence on the moisture content of the cake.

The sheet-like cake thus obtained is dried at a temperature below C ordried at a temperature of l00-l70 C after being pre-dried at atemperature below 80 C. The moisture content of the dried sheet isadjusted thereafter to 10-14 percent (at a temperature of 20 C and ahumidity of 60 percent) in a manner. In the above drying at reducedtemperatures or that with gradually increasing temperature, themembranes of the tobacco cells in the cake are not broken, and thereforea sheet-like material having excellent strength, water-proof propertyand filling capacity can be obtained.

Concerning the reason or mechanism of such improvements in physicalproperties in the products according to the invention, it is consideredthat pectin substance contained in the membranes of the tobacco cellsexists in a so-called physiologically active state and acts as anexcellent binder, and that said pectin substance combines with divalentcations such as calcium, magnesium, and the like, co-existing in saidcake, to become water-insoluble on drying the cake.

Table 2 and 3 illustrate the comparisons of the physical andorganoleptic properties of the sheet-like material, which is produced byusing only the tobacco cells of the present invention, with those ofsheet-like material produced in the conventional extrusion process usingno tobacco cells.

Each test item of the physical properties of the sheetlike materialsshown in Table 2 was measured in the following way:

Thickness (mm) was measured by a micrometer of the dial gauge type at apressure of 550 g./cm

Strength (g./mm was calculated by following the equation which consistsof the above thickness (mm), the below mentioned width (mm) and thetensile load (g.) of the test piece, the tensile load having beenmeasured in such a way that a test piece having a width of 15 mm and alength of 60-70 mm had been tensed (loaded) by using a tensile testerwith constant rate until said test piece was cut.

Strength [Tensile load (g.)]/[Width l 5 mm) X Thickness (mm)] Elongationwas expressed as the percentage of the elongation, which was measured inthe same operation as in the abovementioned measuring of tensile load,based on the length of the test piece not loaded.

Apparent density (g./cm) was calculated from the dried weight (g./cm")and thickness (g.) ofa test piece.

Filling value (g./cigarette) was calculated from the stress measured ona test sample shredded into a width of 0.8 mm and put in a cylinderhaving a cross sectional area of 40 cm and compressed at a rate of 0.6mm/second.

Waterproof property (hours) was expressed as the time needed to brake ofa test piece soaked in water at 25C and in a dish and oscillatedperiodically until the test piece was broken.

The organoleptic properties shown in Table 3 were expressed by thenumber of persons who praised sample cigarettes in the sensory test(applying the pair test method). The test was performed by panelconsisting of ten professional persons who considered smell, taste,mildness and physiological lightness of the cigarettes consisting ofsheet-like materials produced by the present invention and by theconventional extrusion process, respectively.

TABLE 2 Present Conventional Process process Major raw material TobaccoTobacco cells powder Additives Carboxymethyl cellulose Glyoxal 3Physical properties Thickness (mm) 0.20 0. l 3 Strength (gJmm) 300 l54Elongation 5.4 L0 Apparent density (g.lcm") 0.28 0.50 Filling value(gJcigarette) 0.9 1.2 Water-proof property (hrs.) above 50 0.5

TABLE 3 Physiol- Major raw Fla- Taste Mildogical material vour nesslightness Present Tobacco 4 4 l0 l0 process cells Conventional Tobacco 66 0 0 process powder The data in Table 2 show that the sheet-likematerial produced according to the invention is extremely excellentphysical properties in strength, elongation, water-proof property andfilling value, even though no carboxymethylcellulose as a binder andglyoxal as a water-proofing agent were added. Additionally, the data inTable 3 show that the cigarettes which consist of the sheet-likematerial produced according to the invention, are remarkably superior inmildness and physiological lightness, while being almost equivalent inflavor and taste, as compared with those obtained by the conventionalprocess.

FIG. 2 is a schematic flowsheet illustrating an embodiment of the stepsfor producing the sheet-like material of the present invention, where aportion of the cultured broth taken out from fermenter 1 is mixed withaqueous suspension 3 of fibrous materials and/or inorganic materials 2in the mixing tank 4, and after being stirred gently and further passedthrough the reservoir 5, the resulting mixture is supplied onto acontinuously travelling endless gauze 6 at a prescribed rate, so as toform a sheet-like cake having a suitable thickness on said endless gauze6. Said cake is travelled together with the gauze 6 and after tobaccopowder and/or supplementary agents 7 are spread thereon and the cake ispressed by rolls 8, it is stripped off by a doctor blade (not shown) soas to be transferred onto the continuously traveling endless felt 9,which passes through the pre-drying chamber 10 and the main dryingchamber 11. The cake thus having been dried is stripped off by a doctorblade (not shown) to become a sheet-like material for smoking, while theendless felt 9 separated from the sheet-like material is dried ondrum-dryer 12. The filtrate collected in sucking box 13 set under theendless gauze 6 is further separated from a small quantity of solidmatter by the centrifuge l4 and stored transitorily in the tank 15, andis then returned to the initial fermenter 1 after being supplementedwith fresh medium-constituents 16 at the tank 17 and sterilized bysterilizer 18. The solid matter separated from the liquid by thecentrifuge 14 is returned to the mixing tank 4, while a portion of thestored filtrate in tank 15 is preferably returned into the thesuspension 3 so as to improve suspension-abilities of the fibrousmaterials and/or inorganic materials.

According to another embodiment of the present invention, the tobaccocells obtained by the tissue-culture of plants of the genus Nicotianaare made into rodlike material for smoking.

ln the specification of U.S. Pat. No. 3,223,090, it is disclosed that amixture of tobacco powder, water and appropriate additives are cast intorod-shape and freeze-dried so as to form a rod-like material forsmoking.

We have discovered that a rod-like material for smoking similar to thatin the above U.S. Patent can be readily obtained using said tobaccocells. That is, fresh tobacco cells having specific properties ofviscidity and, consequently, a rod-like material having excellentphysical properties can be made by casting the mixture consisting oftobacco cells and appropriate additives into a rod-shape and thereafterfreeze-drying the rodshaped mixture.

According to the present invention, fresh tobacco cells separated fromcultured broth are mixed with tobacco leaf substances and/or thefibrousmaterials and/or inorganic materials which do not damage the smokingcharacteristics of the final products therefrom, to form a paste with amoisture content of -93 percent. lt is preferable to add a small amountof water to the above paste to adjust the moisture content of the paste,when the content of the tobacco cells in the paste is comparativelysmall. The paste thus formed can be readily cast into a rod-shape, andthereafter freeze-dried so as to be set in substantially the same formas when cast. On the contrary, a paste with moisture content of below 80percent or above 93 percent is unsuitable for being physically cast intoa rodshape.

ln the present invention, the supplementary agents such as thearomatics, sweetenings, hydroscopic agents and burning agents, which areused generally in the production of conventional re-constituted tobacco,may be added to the said mixture of tobacco cells with tobacco leafsubstances and/or the fibrous materials and/or inorganic materials.However, the aromatics, sweetenings and hydroscopic agents among saidsupplementary agents may be later incorporated into the finished(freeze-dried) rod-like material in the invention. The kinds of fibrousmaterials and inorganic materials which do not damage the smokingcharacteristics of the final product and the supplementary agents whichmay be used in the present invention, are the same as describedpreviously.

The tobacco leaf substances and/or the fibrous materials and/orinorganic materials to be mixed with the tobacco cells are effective thephysical and organoleptic properties of the dried rod-like material, andparticularly strength and filling value. The quantities of suchadditives are from 0.3- to 2.0-fold of the dried weight of the tobaccocells used. The quantities of the supplementary agents to be added arerather small, and therefore addition of these agents have littleinfluence on the moisture content of the said paste.

Table 4 illustrates the relationship between the composition andmoisture content of a pasteconsisting of tobacco cells with a moisturecontent of 93 percent and dried tobacco powder.

TABLE 4 Composition (Dried weight Moisture content In order to cast theabove paste into a rod-shape, continuous cylindrical extrusion-machinesare preferably used. However, the paste may be frozen as is in a casthaving a rod-shape. Boring fine holes of from 0.2 mm to 0.5 mm diameter,lengthwise through the rod-shaped paste improves the draw resistance insmoking of the final product.

The paste, cast into rod-shape is then flash-frozen by placing the samein a zone at a temperature of from about 20 to 80 C, and is thereafterfreeze-dried at a temperature of less than 5 C and at a pressure of lessthan 3 mmHg. If the paste is frozen slowly, large crystals of ice areformed in the frozen rod, and this is disadvantageous that the dryingtime is prolonged and the inner pores of the finished rod-like materialbecome larger and less uniform.

1n the above freeze-drying, the moisture (ice) of the frozen paste isremoved by sublimation, and consequently the frozen paste is driedwithout any shrinkage, and the original shape and size are maintained.Since the fresh tobacco cells used as the raw material in the inventioncontain generally moisture of more than 90 percent (W.B.) inside thecell-membranes thereof, the freeze-dried rod-like material therefrom hasinnumerable fine pores and remarkably little apparent density. Further,as the fresh tobacco cells are cohesive and act as a binder in thepaste, the tobacco leaf substances and other additives in the driedrod-like material are incorporated together strongly by the tobaccocells, while the rod acquires appropriate elasticity.

The rod-like material obtained above, after the moisture content isadjusted to -14 percent, and upon being encased as it is in a wrapper orfurther attached with filter tip at one end is ready for sale andsmoking. This cigarette has mild and light organoleptic properties dueto the low content of nicotine and tar, and has good burning-ability dueto the many inner pores.

Table 5 shows examples of the equilibrium moisture content (W.B.) of therod-like material made by freezedrying a paste consisting of tobaccocells and tobacco powder, both derived from N. tabacum var. brightyellow. The equilibrium moisture content was measured on a sample placedin a sulfuric acid-desiccator at a temperature of C the belowmentionedhumidities. After attaining a constant moisture content, the sampleswere dried at a temperature of 100 C for l hour.

TABLE 5 Tobacco Tobacco Tobacco Tobacco Composition cells cells cellspowder (dried weight) 100 69 51 100 Tobacco Tobacco Relative powderpowder humidity 31 49 50% 13.0% 11.5 10.8 9.0% 60 16.1 14.7" 13.7 11.570 21.0 18.9 18.0 14.9 80 29.5 27.1 25.7 21.8

The following examples serve to illustrate the invention without howeverlimiting it:

EXAMPLE 1 A seed of N. tabacum var. bright yellow was washed withdeionized water, soaked in percent ethanol for several seconds, furthersoaked in 10 percent sodium hypochlorite solution for about 10 minutesand thereafter washed with sterile water. Said seed was placed on asterilized solid culture-medium, which is prepared by adding 1 percentof agar to the composition shown in Table 1 put in a cotton-pluggedErlenmeyers flask, and incubated at a temperature of 26-3O C for about 4weeks. When a budding portion of the seed was brought in contact withthe solid medium, callus was derived from that contact point. Thiscallus was cut off, transferred onto a fresh medium having the samecomposition as the above, and cultured. After such a sub-culture ofcallus had been repeated thrice, about 3 g. of the callus by freshweight formed in the last culturing were inoculated into ml. of liquidmedium of the same composition but without agar held in a 500ml.-Sakaguchis flask, and cultured on a shaker at a temperature of 2830C. After about 2 weeks, tobacco cells were propagated in suspension inthe liquid. About 10 ml, of this cultured broth were transferred into100 ml. of fresh liquid medium of the same composition andshake-cultured again. After such a sub culture had been repeated fivetimes, cultured broth, where tobacco cells were more finely anduniformly dispersed, was obtained in about a week.

About 100 ml. of said cultured broth were then in oculated into 1 literof liquid medium of the same composition held in a 3 liter-flask andshake-cultured for a week. The cultured broth obtained in the above 3literflask was inoculated into 11 liters of liquid medium held in a 15liter-jar-fermenter, and cultured at a temperature of 30 C, under anaeration rate of 7.5 liters/minute and an agitating rate of 50 rpm.After 56 days, about 5 liters, that is, about a half of the culturedbroth in the jar-fermenter where tobacco cells had been propagated insuspension, were taken out and about 5.5 liters of fresh medium, whichhad been separately prepared and sterilized, were supplemented to thefermenter and aeration-culture with agitating was carried on under thesame conditions as described above except that the aeration rate wasincreased to 11.5 liters/minute. After 2 days about 5 liters of culturedbroth were similarly taken out of the fermenter and fresh medium wassupplemented to the fermenter again. Thus, tobacco cells of 131 5 g. bydried weight per liter of the cultured broth were obtained every otherday, by repeating the abovementioned semi-continuous culture of thetobacco cells.

10 liters of cultured broth containing 142 g. of the dried weight oftobacco cells prepared as described above were filtered by means ofsuction through a gauze of 100 mesh, so as to form a sheet-like cake onthe gauze, the dried weight/m of which was 80 g. three grams of sorbitoland a small quantity of cascarilla oil were made to stick on the cake byspreading the same and pressing such cake gently between rolls. Thepressed cake was pre-dried by using a hot air stream at a temperature of60 C to a moisture content of about 60 percent (W.B.), and then dried byusing hot air stream at a temperature of 100 C to a moisture contentbelow percent. Thereafter, the moisture content of the dried sheet wasadjusted to 12 percent (at a temperature of 20 C and a humidity of 60percent).

The thus obtained sheet-like material had a strength of 300 g./mm anapparent density of 0.28 g./cm*, a filling value of 0.89 g./cigarette, awater-proof property of at least 50 hours, and the cigarettes made byusing this sheet were superior in mildness and physiological lightness.

EXAMPLE 2 Tobacco cells were propagated by a similar operation to thatin Example 1 except that a fragment of a leaf of N. tabacum var. xanthiwas used instead of a seed of N. t. var. bright yellow. Fifteen grams ofwood pump (N-BKP) dispersed in a hundred-fold quantity of water wereadded to 10 liters of cultured broth (containing 145 g. of tobacco cellsby dried weight), and the resulting mixed liquid was filtered by meansof suction through a filter cloth, so as to form a sheet-like cakehaving a thickness corresponding to dried weight of 70 g./m This cakewas a dried by using a hot air stream at a temperature of 70 C to amoisture content below 10 percentaTwo grams of sugar and a smallquantity of benzoin tincture were sprinkled on the dried cake andsimultaneously the moisture content of the cake was adjusted to 12percent.

The thus obtained sheet had a strength of 450 g./mm a water-proofproperty of at least 50 hours, a filling value of 0.70 g./cigarette, andthe cigarettes made by using said sheet were superior in mildness andphysiological lightness.

EXAMPLE 3 Tobacco cells were propagated by a similar operation to thatin Example 1 except that a fragment of root of N. glutinosa was usedinstead of a seed of N. t. var. bright yellow. Ten liters of culturedbroth (containing 142 g. of tobacco cells by dried weight), were mixedwith 30 g. of vein of tobacco leaf which had been refined and dispersedin a 25-fold quantity of water. The mixed dispersion liquid was filteredand made into a sheet-like material in the same way as in Example 1,except that 4 g. of glycerin and a small quantity of bergamot oil wereused as supplementary agents.

The thus obtained sheet had a strength of 400 g./mm at least water-proofproperty of at least 50 hours, a filling value of 0.70 g./cigarette, andthe cigarettes made therefrom were superior in mildness andphysiological lightness, while being excellent in flavor and taste.

EXAMPLE 4 Tobacco cells were propagated in the same way as in Example lexcept that a fragment of the stem of N. debneyi was used instead of aseed of N. 1. var. bright yellow. Ten liters of the cultured broth(containing 137 g. of tobacco cells by dried weight), were mixed with 15g. of calcium carbonate and I0 g. of wood pulp which had been dispersedin a 50-fold quantity of water. The resulting dispersion liquid wasfiltrated and made into a sheet-like material in the same way as inExample 1 except that 3 g. of propyleneglycol and a small quantity ofiris oil there used as supplementary agents:

The thus obtained sheet had a strength of 470 gJmm, an apparent densityof 0.30 g./cm", a filling value of 0.65 g./cigarette, a water proofproperty of at least 50 hours, and the cigarettes made therefrom weresuperior in mildness and physiological lightness.

EXAM PLE 5 Tobacco cells were progagated in the same way as in Example 1except that a fragment of bud of tabacum var. nambu was used instead ofa seed of N. t. var. bright yellow. 10 liters of the cultured broth(containing 141 g. of tobac:o cells by dried weight) were mixed with 12g. of glass fiber, and the resulting dispersion was filtered and madeinto a sheet-like material in the same way as in Example 1 except that 3g. of sorbitol and a small quantity of tobacco extract were used assupplementary agents.

The thus obtained sheet had a strength of 420 g./mm a water-proofproperty of at least 50 hours, a filling value of 0.68 g./cigarette, andthe cigarettes made therefrom were superior in mildness andphysiological lightness.

EXAMPLE 6 Tobacco cells were propagated in the same way as in Example 1except that a fragment of a petal of N. tabacum var. enshu was usedinstead of a seed of N. 1. var. bright yellow and that the filtrate ofthe cultured broth taken from the jar-fermenter was returned to thejar-fermenter after being supplemented with fresh medium-constituentsand sterilized instead of the fresh medium which had been newly preparedand supplemented into the jar-fermenter. Ten liters of the culturedbroth (containing 144 g. of tobacco cells by dried weight) were filteredin the same way as in Example l, and g. of tobacco powder, 3 g. ofpropyleneglycol and a small quantity of iris oil was made to stick onthe cake by spreading the same and pressing the cake between rolls. Theresulting cake was dried in the same way as in Example 2 and thereafterthe moisture content thereof was adjusted to 13 percent.

The thus obtained sheet had a strength of 2l0 g./mm", a water proofproperty of at least 50 hours, a filling value of 0.95 g./cigarette, andthe cigarettes made therefrom were superior in mildness andphysiological lightness, while being excellent in flavor and taste.

EXAMPLE 7 Tobacco cells were propagated in the same way as in Example 1and separated from the culturedbroth by means of a centrifuge.

l.5 kg. of the tobacco cells by fresh weight (containing l03 g. by driedweight) collected from the above were mixed and kneaded with l00 g. ofparticles having a size corresponding to meshes of l6-32 prepared bydrying and grinding shredded tobacco obtained from cigarettes marketedunder the trademark hi-lite," l0 g. of sorbitol and a small quantity ofcascarilla oil, so as to form a paste with a moisture content of 87percent (W.B.). The paste was cast using a cylindrical extrusion-machineinto a rod-shape having a diameter of 8 mm and a length of 63 mm, whilesimultaneously boring the rod with four fine holes with a diameter of0.5 mm lengthwise, by using thin wire cores. The rodshaped paste wasflash-frozen by placing the same in a zone at a temperature of 20 C.Thereafter the frozen rod was freeze-dried by means of a vacuumfreeze-drier at a pressure of 2.5 mmHg and a temperature of -7.2 C. Thedried rod-like material had innumerable fine pores inside. After themoisture content of this rod had been adjusted to 13 percent, said rodwas encased in a wrapper together with a filter tip of length having a17 mm attached to one end of the rod, and submitted to physicalpropertyand smoking-tests to compare the properties with those of thegoods on the market (trademark hi-lite"). The results are shown in Table6.

Table 6 Goods on the market (hi-lite) Products of the invention Fillingvalue (gJcigarette) 0.47 0.85 Hardness (kg/cigarette) 1.50 0.87 Nicotinein smoke (mg/cigarette) 0.3 1.7 TPM in smoke 9.0 27.9 Draw resistance insmoking (mmH O) 1 108 Each test item in the Table 6 was measured in thefollowing way:

Filling value was measured in the same way as described previously.

Hardness was expressed as'stress caused by pressing thetest cigarette inits diameter-direction until distortion of 1.5 mm occurred.

TPM in smoke (Total Particulate Matter in smoke) was expressed as theweight increase of an aerosol filter (Glass Fiber Sheet CM 113A, made byCambridge Filter Co., Ltd.) caused by adhesion of crude tar (moist tar)on the filter, when smoke of the test cigarette was drawn through saidfilter.

Nicotine in smoke was calculated from the wave length of ultravioletabsorption of an aqueous solution which had been prepared as followscrude tar adhered onto the aerosol filter in the abovementionedmeasuring of TPM in smoke was steam-distillated in acidic condition, andthen the residue thereof was steamdistillated again but in alkalinecondition, and thereafter the distillate was acidified.

Draw resistance of the rod in smoking was expressed as the pressuredifference indicated by a U-tube manometer filled with water when an airstream was drawn at a rate of 17.5 ml./second by means of a vacuum pumpthrough the test cigarette and the U-tube manometer in parallel, under atemperature of C and a relative humidity of 60 percent.

As seen from the results of Table 6, the rod-like material produced inthe present invention is superior in filling value, quantities ofnicotine and TPM in smoke as compared with the goods on the market,while being slightly higher in draw resistance.

EXAMPLE 8 Tobacco cells were propagated and collected in the same way asin Example 7 except that a fragment of a leaf of N. 1. var. xanthi wasused instead ofa seed of N. 1. var. bright yellow. 1.5 kg. of thetobacco cells by fresh weight (containing 105 g. by dried weight)collected were mixed and kneaded with 75 g. of particles having a sizecorresponding to meshes of 1632 prepared by drying and grinding shreddedtobacco from cigarettes marketed under the trademark RAN", g. of calciumcarbonate, 5 g. of sugar and a small quantity of water, so as to form apaste with a moisture content of percent (W.B.). The paste was filledinto a pipe having a diameter of 8 mm and a length of 63 mm, whilesimultaneously boring the paste with four fine holes, each with adiameter of 0.5 mm, by passing thin wire cores intothe pipe. The pastewas flash-frozen as is by placing the same in a zone at a temperature of20 C. The frozen rod was then freeze-dried by means of a vacuumfreeze-drier at a pressure of 3 mmHg and at a temperature of 5 C. Asmall quantity of iris oil was incorporated into the dried rod-likematerial, and simultaneously the moisture content of the rod wasadjusted to l 1 percent. The rod was then encased with a wrapper in thesame way as in Example 7. This cigarette was submitted to physicalpropertyand smoking to compare its properties with those of the goodson. The market (trademark RAN). The results are shown in Table 7.

EXAMPLE 9 Tobacco cells were propagated and collected in the same way asin Example 7 except that a fragment of a root of N. glutinosa was usedinstead of a seed of N. 1. var. bright yellow. 1.5 kg. of the tobaccocells by fresh weight (containing 102 g. by dried weight) collected weremixed and kneaded with g. of particles having a size corresponding tomeshes of 16-32 prepared by drying and grinding shredded tobacco fromcigarettes marketed under the trademark Winston, 10 g. of wood pulp(N-BKP) and a small quantity of tobacco extract, so as to form a pastewith a moisture content of 86 percent (W.B.). The paste was thereaftercast into rod-like material for smoking in the same way as in Example 7.

EXAMPLE 10 The tobacco cells were propagated and collected in the sameway as in Example 7 except that a fragment of a stem of N. debneyi wasused instead of a seed of N. t. var. bright yellow. 1.5 kg. of tobaccocells by fresh weight (containing 104 g. by dried weight) were mixed andkneaded with 100 g. of particles having a size corresponding to meshesof 1632 prepared by drying and grinding shredded tobacco from cigarettesmarketed under the trademark LARK," 10 g. of propyleneglycol and a smallquantity of benzoin tincture, so as to form a paste containing amoisture content of 87 percent (W.B.). The paste was thereafter castinto rod-like material for smoking in the same way as in Example 7.

EXAMPLE 11 Tobacco cells were propagated and collected in the same wayas in Example 7 except that a fragment of a bud of N. tabacum var. nambuwas used instead of a seed of N. t. var. bright yellow. 1.5 kg. oftobacco cells by fresh weight (containing 106 g. by dried weight) weremixed and kneaded with 100 g. of particles having a size correspondingto meshes of 16-32 prepared by drying and grinding shredded tobacco fromthe cigarettes marketed under the trademark ROTHMANSf 5 g. of activecarbon and a small quantity of menthol, so as to form a paste with amoisture content of 88 percent (W.B.). The paste was thereafter castinto rod-like material for smoking in the same way as in Example 7.

EXAMPLE 12 Tobacco cells were propagated in the same way as in Example6. 1.5 kg. of tobacco cells by fresh weight (containing 101 g. by driedweight) collected were thereafter treated in the same way as in Example7, and rod-like material for smoking was obtained.

What we claim is:

1. A process for producing sheet-like material for smoking, whichcomprises:

1. culturing a fragment of a plant body of the genus Nicotiana on asolid agar medium for plant tissueculture and further comprising amixture of growth-promoting agents selected from the group consisting ofsaccharides, inorganic salts, auxins, cytokinins and vitamins, to derivean amorphous lump of cells, designated as callus, on said solid medium,

. inoculating said callus into a liquid medium of the same compositionas described above, and culturing the same under aerobic conditions, soas to propagate it into a state of tobacco cells suspended in theliquid,

3. filtering the cultured liquid containing the tobacco cells through agauze of 50-120 meshes or filter cloth, so as to form a sheet-like cakehaving a thickness corresponding to a dried weight above 50 g./m and 4.drying the sheet-like cake.

2. The process described in claim 1, where the cultured liquid isfiltered so as to form a sheet-like cake, after said liquid haspreviously been mixed with at least one member selected from the groupconsisting of fibrous materials and inorganic materials, said fibrousmaterials and inorganic materials not damaging the smokingcharacteristics of the final product thereof.

3. The process described in claim 1, where the sheetlike cake is dried,after at least one member selected from the group consisting of tobaccoleaf substances and supplementary agents has previously been made tostick on the sheet-like cake.

4. The process described in claim 2, where the fibrous material is afiber selected from the group consisting of wood pulp, hemp, bagasse,beet, carbon fiber, and tobacco vein.

5. The process described in claim 2, where the inorganic material issilica, alumina, asbestos, kieselguhr, zeolite, talc or calciumcarbonate.

6. The process described in claim 3, where the supplementary agent is atleast one member selected from the group consisting of aromatics,burning agents,

sweetenings and hydroscopic agents.

7. The process described in claim 2, where at least one member selectedfrom the group consisting of fibrous materials and inorganic materialsis used in an amount below 30 percent based on the dried weight of thetobacco cells contained in the sheet-like cake.

8. The process described in claim 3, where the tobacco leaf substancesare used in a quantity below the value of the dried weight of thetobacco cells contained in the cake.

9. The process described in claim I, where the sheetlike cake is driedat a temperature below C.

10. The process described in claim 1, where the sheet-like cake ispre-dried at a temperature below 80 C, and is thereafter dried at atemperature of l00-l70 11. The process described in claim 1, where atleast one member selected from the group consisting of aromatics,sweetenings and hydroscopic agents is sprinkled onto the driedsheet-like cake. 1

12. The process described in claim 1, where the inorganic phosphate inthe medium for plant tissue-culture is used in a quantity from twice tothrice as much as that in the conventional Linsmaier and Skoogs medi-13. A process for producing rod-like material for smoking whichcomprises:

1. culturing a fragment of a plant body of the genus Nicotiana on asolid agar medium for plant tissueculture and further comprising amixture of growth-promoting agents selected from the group consisting ofsaccharides, inorganic salts, auxins, cytokinins and vitamins, to derivean amorphous lump of cells, designated as callus, on said solid medium,

2. inoculating said callus into a liquid medium of the same compositionas described above, and culturing the same under aerobic conditions, soas to propagate it into a state of tobacco cells suspended in theliquid,

3. separating the tobacco cells from the cultured liquid,

. mixing the tobacco cells with at least one member selected from thegroup consisting of tobacco leaf substances, fibrous materials andinorganic materials, to form a paste with a moisture content of 80-93percent, said fibrous materials and in'organic materials not damagingthe smoking characteristics of the final product thereof,

5. casting the past into rod-shape, and

6. freeze-drying said rod-shaped paste.

14. The process described in claim 13, where the paste is cast into arod-shape, said paste having previ' ously been mixed with supplementaryagents.

15. The process described in claim 13, where the fibrous material is afiber selected from the group consisting of wood pulp, hemp, bagasse,carbon fiber and beet.

16. The process described in claim 13, where the inorganic material issilica, alumina, asbestos, kieselguhr, zeolite, talc or calciumcarbonate.

17. The process described in claim 14, where the supplementary agent isat least one member selected from the group consisting of sweetenings,hydroscopic agents, burning agents and aromatics.

18. The process described in claim 13, where at least one memberselected from the group consisting of tobacco leaf substances, fibrousmaterials and inorganic materials is used in an amount from 0.3- to 2.0-fold of the dried weight of the tobacco cells used.

19. The process described in claim 13, where at least one memberselected from the group consisting of aromatics, sweetenings andhydroscopic agents is sprin-

2. inoculating said callus into a liquid medium of the same compositionas described above, and culturing the same under aerobic conditions, soas to propagate it into a state of tobacco cells suspended in theliquid,
 2. inoculating said callus into a liquid medium of the samecomposition as described above, and culturing the same under aerobicconditions, so as to propagate it into a state of tobacco cellssuspended in the liquid,
 2. The process described in claim 1, where thecultured liquid is filtered so as to form a sheet-like cake, after saidliquid has previously been mixed with at least one member selected fromthe group consisting of fibrous materials and inorganic materials, saidfibrous materials and inorganic materials not damaging the smokingcharacteristics of the final product thereof.
 3. The process describedin claim 1, where the sheet-like cake is dried, after at least onemember selected from the group consisting of tobacco leaf substances andsupplementary agents has previously been made to stick on the sheet-likecake.
 3. separating the tobacco cells from the cultured liquid, 3.filtering the cultured liquid containing the tobacco cells through agauze of 50-120 meshes or filter cloth, so as to form a sheet-like cakehaving a thickness corresponding to a dried weight above 50 g./m2, and4. drying the sheet-like cake.
 4. mixing the tobacco cells with at leastone member selected from the group consisting of tobacco leafsubstances, fibrous materials and inorganic materials, to form a pastewith a moisture content of 80-93 percent, said fibrous materials andinorganic materials not damaging the smoking characteristics of thefinal product thereof,
 4. The process described in claim 2, where thefibrous material is a fiber selected from the group consisting of woodpulp, hemp, bagasse, beet, carbon fiber, and tobacco vein.
 5. Theprocess described in claim 2, where the inorganic material is silica,alumina, asbestos, kieselguhr, zeolite, talc or calcium carbonate. 5.casting the past into rod-shape, and
 6. freeze-drying said rod-shapedpaste.
 6. The process described in claim 3, where the supplementaryagent is at least one member selected from the group consisting ofaromatics, burning agents, sweetenings and hydroscopic agents.
 7. Theprocess described in claim 2, where at least one member selected fromthe group consisting of fibrous materials and inorganic materials isused in an amount below 30 percent based on the dried weight of thetobacco cells contained in the sheet-like cake.
 8. The process describedin claim 3, where the tobacco leaf substances are used in a quantitybelow the value of the dried weight of the tobacco cells contained inthe cake.
 9. The process described in claim 1, where the sheet-like cakeis dried at a temperature below 80* C.
 10. The process described inclaim 1, where the sheet-like cake is pre-dried at a temperature below80* C, and is thereafter dried at a temperature of 100*-170*C.
 11. Theprocess described in claim 1, where at least one member selected fromthe group consisting of aromatics, sweetenings and hydroscopic agents issprinkled onto the dried sheet-like cake.
 12. The process described inclaim 1, where the inorganic phosphate in the medium for planttissue-culture is used in a quantity from twice to thrice as much asthat in the conventional Linsmaier and Skoog''s medium.
 13. A processfor producing rod-like material for smoking which comprises:
 14. Theprocess described in claim 13, where the paste is cast into a rod-shape,said paste having previously been mixed with supplementary agents. 15.The process described in claim 13, where the fibrous material is a fiberselected from the group consisting of wood pulp, hemp, bagasse, carbonfiber and beet.
 16. The process described in claim 13, where theinorganic material is silica, alumina, asbestos, kieselguhr, zeolite,talc or calcium carbonate.
 17. The process described in claim 14, wherethe supplementary agent is at least one member selected from the groupconsisting of sweetenings, hydroscopic agents, burning agents andaromatics.
 18. The process described in claim 13, where at least onemember selected from the group consisting of tobacco leaf substances,fibrous materials and inorganic materials is used in an amount from 0.3-to 2.0-fold of the dried weight of the tobacco cells used.
 19. Theprocess described in claim 13, where at least one member selected fromthe group consisting of aromatics, sweetenings and hydroscopic agents issprinkled onto the dried rod-like material.
 20. The process described inclaim 13, where the inorganic phosphate in the medium for planttissue-culture is used in a quantity from twice to thrice as much asthat in the conventional Linsmaier and Skoog''s medium.